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Neurotransmitters regulate neuronal function in the nervous system and modulation of their synthesis, release, and binding by immature neurons and their targets is a major part of nervous system development. We propose that the neurotransmitter noradrenaline regulates neuronal fate during neurulation, before neurons have differentiated. The ability of noradrenaline to induce a neural fate was tested in naive ectoderm caps cut from late blastula stage Xenopus embryos. Noradrenaline (10(-6) M) did not switch on otx-2 or NCAM and did not induce the formation of cement glands. We conclude that noradrenaline cannot induce a neural fate. By contrast, 10(-8) M noradrenaline activated N-tubulin in ectoderm caps expressing the neural inducing molecule noggin by the time intact siblings had become mid-neurulae. Methoxamine, a specific alpha-adrenergic receptor agonist, also activated N-tubulin in noggin-expressing caps. The alpha-adrenergic receptor blocker prazosin inhibited both noradrenaline- and methoxamine-induced activation of N-tubulin. The neurotransmitters dopamine and 5-HT did not activate expression of N-tubulin. XA-1, Otx-2, X-Delta, and Xotch transcripts were not sensitive to noradrenaline. HoxB9, which indicates posteriorization, was not activated by noradrenaline. When intact siblings were at stage 27, many cells in noggin-expressing, noradrenaline-treated caps were stained by the neuron-specific mcAb3A10. We propose that noradrenaline is an important endogenous modulator of neuronal fate, driving noggin-expressing cells to become neurons by binding to alpha-adrenergic receptors and activating a cascade that culminates in the expression of the neuronal markers N-tubulin and 3A10.
FIG. 1. Noradrenaline is not a primary neural inducer. Ectoderm caps from late blastula stage embryos treated with noradrenaline and
assayed at stage 17 (A) or stage 12/13 (B, C). E, intact embryos. C, ectoderm caps. NA, ectoderm caps treated with noradrenaline. n,
noggin-expressing caps. n/NA, noggin caps treated with noradrenaline. (A) Noradrenaline does not induce expression of NCAM in naive
ectoderm caps. (B) NA induces XA-1, but this is not prevented by prazosin. (C) NA does not induce Otx-2.
FIG. 2. Noradrenaline induction of N-tubulin in noggin-expressing caps is prevented by the a-adrenergic receptor blocker prazosin. NA
(102 6 M): (A) stage 30, (B) stage 17. (C) NA (1027 M), stage 17. (D) Doseâresponse relationship for induction of N-tubulin by noradrenaline.
Note that 1028 MNA is sufficient.
FIG. 3. 5-HT (A) and dopamine (B) do not induce N- tubulin. The
a-adrenergic agonist methoxamine (M) induces N-tubulin and this
induction is blocked by prazosin (C).
FIG. 4. Noradrenaline does not influence expression of Xotch (A),
X-Delta (B), or HoxB9 (C).
FIG. 5. At stage 17, N-tubulin-expressing cells are not uniformly distributed in noggin-expressing, noradrenaline-treated ectoderm
caps. (A) noggin-expressing ectoderm caps do not express N-tubulin. (B, C) Noradrenaline (1026 M) induces expression of N-tubulin. Cells
in the outer layer do not express N-tubulin and N-tubulin-expressing cells are not distributed uniformly. The high-power image in C shows
that N-tubulin-expressing cells are interspersed with nonexpressing cells. Bar 5 160 mm for A and B and 480 mm for C.
FIG. 6. At stage 27, noradrenaline-treated noggin caps contain many N-tubulin-expressing cells. (AâC) noggin caps treated with 1026 M
noradrenaline. (A) Low-power image to show typical internal organization of Na-treated caps. The N-tubulin-expressing cells are concentrated
in one region of the cap. The cement gland (arrowed) lies to one side, in a region where the majority of cells do not express N-tubulin. (B, C)
Sections through two other 1026 MNa-treated noggin caps photographed at higher power to show the organization of N-tubulin-expressing cells.
Note that even in regions where N-tubulin-expressing cells are at high density, not all cells express N-tubulin. (D) noggin cap treated with 1026
MNA and prazosin. This is an example of a cap where prazosin abolished completely the ability ofNAto induce N-tubulin expression. (E) noggin
cap that had not been treated with NA. Untreated noggin caps never contained N-tubulin-positive cells. The scattered dark cells apparent in D
and E are pigmented. (F) High-power image to show the stellate shape of N-tubulin-expressing cells (arrow) within the noradrenaline-treated cap.
Bar 5 200 mm for A, D, and E, 125 mm for B and C, and 40 mm for F.
FIG. 7. mcAb3A10 staining reveals that by stage 27 noradrenalinetreated,
noggin-expressing caps contain neurons. (A, B, C) noggin caps
treated with 1026 M noradrenaline. 3A10-positive cells are present at
high density with many short neurites. In A and C the region
containing most neurites lies between the arrows. (D) noggin cap
treated with 1027 Mnoradrenaline. The density of 3A10-positive cells
is reduced, although neurites are clearly visible (arrow). A substantial
proportion of the cap does not contain 3A10-positive cells. (E) Part of
noggin cap treated with 1026 M noradrenaline and prazosin. The only
3A10-positive cells are present in a small part of the cap (arrows). (F)
A noggin-expressing cap that had not been treated with noradrenaline.
3A10-positive cells are absent. Each image shows a projection through
the frozen section constructed from six stacked 2-mmoptical sections.
Bar 5 100 mm. (G) Neural tubemarginal zone in an intact Xenopus
embryo stained with 3A10. Note dense neurite staining with some
neurites coursing into surrounding tissues (e.g., arrows). Single 4-mm
optical section. Bar 5 50 mm.
